Alternative splicing of the Sex-lethal pre-mRNA has long served as a model example of a regulated splicing event, yet the mechanism by which the female-specific Sex lethal RNA-binding protein prevents inclusion of the translation-terminating male exon is not understood. Thus far, the only general splicing factor for which there is in vivo evidence for a regulatory role in the pathway leading to male-exon skipping is Sans-fille (Snf), a protein component of the spliceosomal U1 and U2 snRNPs. Its role, however, has remained enigmatic because of questions about whether Snf acts as part of an intact snRNP or a free protein. Evidence is provided that Sex lethal interacts with Sans-Fille in the context of the U1 snRNP, through the characterization of a point mutation that interferes with both assembly into the U1 snRNP and complex formation with Sex lethal. Moreover, Sex lethal associates with other integral U1 snRNP components, and genetic evidence is provided to support the biological relevance of these physical interactions. Similar genetic and biochemical approaches also link Sex lethal with the heterodimeric splicing factor, U2AF (see U2 small nuclear riboprotein auxiliary factor 50). These studies point specifically to a mechanism by which Sex lethal represses splicing by interacting with these key splicing factors at both ends of the regulated male exon. Moreover, because U2AF and the U1 snRNP are only associated transiently with the pre-mRNA during the course of spliceosome assembly, these studies are difficult to reconcile with the current model that proposes that the Sex lethal blocks splicing at the second catalytic step, and instead argue that the Sex lethal protein acts after splice site recognition, but before catalysis begins (Nagengast, 2003).

The Sxl male exon is unusual in that it contains two 3' AG
dinucleotides separated by a short polypyrimidine tract.
Interestingly, although the upstream 3' splice site is used almost
exclusively for exon ligation in tissue-culture cells, both 3' splice
sites are required for Sxl-mediated male-exon skipping.
Moreover, crosslinking studies in HeLa cell extracts have shown that the U2AF
heterodimer binds to the downstream 3' splice site and the intervening
polypyrimidine tract, suggesting that U2AF may play an active role in
Sxl regulation. These biochemical data have been validated by demonstrating
that the Sxl protein can associate with the Drosophila U2AF
orthologs. More importantly, genetic data provide compelling support for
the biological relevance of these interactions by demonstrating that in
females, the small subunit is important for both Sxl male-exon
skipping and female viability. In addition to demonstrating a role for U2AF in
Sxl autoregulation, this genetic result is notable because previous
studies have failed to find RNA splicing defects associated with small subunit
mutations. Whether this success reflects substrate-specificity or
sensitivity of the assay remains to be determined (Nagengast, 2003).

In addition to controlling the use of the male exon 3' splice site,
these studies suggest that Sxl controls the use of the male-exon 5' splice
site by interacting with the U1 snRNP. This
connection was established in three ways: (1) it was found that mutation of a single residue in
the N-terminal RRM of SNF compromises both complex formation with Sxl and
assembly into the U1 snRNP, thus suggesting that the two events are linked; (2) it has been demonstrated that, in addition to SNF, Sxl can associate with other
integral U1 snRNP components, including the U1-70K protein and the U1 snRNA in
whole cell extracts; (3) genetic interaction data provide evidence
that U1-70K, like SNF, is important for the successful establishment of the
Sxl autoregulatory splicing loop in females (Nagengast, 2003).

Although the discovery that SNF is an snRNP protein was the first clue that
Sxl might act by associating with components of the general splicing
machinery, the role of SNF has remained enigmatic. The role of SNF has been clarified
by demonstrating that its contribution to the function of the U1 snRNP is not
absolutely essential for viability of either sex, and that Sxl can associate
with the U1 snRNP through a SNF-independent mechanism. Nevertheless, in
vivo analysis continues to support a role for snf in Sxl
splicing autoregulation by demonstrating that Sxl splicing defects
are detectable under specific conditions. Interestingly, the phenotypic
consequences of these Sxl splicing defects are more severe in the
germline than in the soma. One possible explanation for this difference is
that the requirements for Sxl splicing autoregulation are
fundamentally different in the two tissue types. It is thought
more likely that the mechanism is the same, but that the additional
interaction with the U1 snRNP provided by SNF becomes critical when Sxl
protein levels are low. This hypothesis is based on the fact that, in the
germline, the majority of Sxl protein is cytoplasmic, and thus low levels of
nuclear Sxl protein are the norm. By contrast, in other tissues, the Sxl protein
accumulates in the nucleus, enabling the Sxl-U1 snRNP complex to form even
when SNF is not stably associated with the U1 snRNP. The finding that these
snf mutant females rarely survive if they are also heterozygous for
Sxl, provides additional support for the idea that SNF function is
only critical when Sxl protein levels are low (Nagengast, 2003).

Together, these studies argue that interactions between Sxl, the U1 snRNP and
U2AF underlie the mechanism by which Sxl promotes skipping of the male exon.
Based on these studies, a model is proposed in which Sxl acts not by preventing
assembly of the U1 snRNP or U2AF onto the pre-mRNA, but instead interacts with
the U1 snRNP bound to the male-exon 5' splice site, and U2AF at the
male-exon 3' splice site, to form complexes that block these general
splicing factors from assembling into a functional spliceosome. These 5' and
3' Sxl blocking complexes might function independently or they might
interact across the exon to form a larger inhibitory complex. Furthermore,
because it has not been possible to demonstrate that Sxl interacts directly with
either U1-70K or U2AF, it is speculated that one or more bridging proteins are
required to link Sxl to the general splicing machinery (Nagengast, 2003).

Although the in vivo approach cannot directly address when in the pathway
of spliceosome assembly Sxl acts, biochemical studies have shown that during
the course of spliceosome assembly, U2AF and the U1 snRNP are only transiently
associated with splicing substrates, and are released before the formation of
a functional spliceosome. Therefore, based on these studies, it seems reasonable
to propose that Sxl acts by blocking splicing after splice site recognition
but before catalysis begins. The data are therefore difficult to reconcile
with the recent model, which
proposes that Sxl blocks splicing after spliceosome assembly, at the second
catalytic step of the reaction. Using RNA interference in Drosophila
tissue culture cells it has been
demonstrated that efficient male exon skipping depends on the presence of
SPF45, a protein that is known to be required for the second step of splicing.
Together with studies that show that SPF45 can bind to the upstream 3'
splice site of the Sxl male exon and physically interact with Sxl,
these data point to a role for SPF45 in Sxl splicing regulation.
However, the primary evidence that Sxl blocks the splicing reaction during the
second step rests on the results of in vitro splicing assays in which Sxl was
shown to inhibit splicing of a chimeric splicing substrate that contains only
a small region of the intronic region required for successful autoregulation
in vivo. It is suspected that by looking at this 48 bp region, which
contains a dispensable Sxl-binding site in addition to the two potential
3' splice sites, out of context, a failsafe mechanism was uncovered that comes into play when Sxl-mediated splicing
regulation is otherwise compromised. Additional studies investigating the
function of SPF45 in vivo will be required to determine the importance of this
second step blocking mechanism and should provide insight into whether
multiple mechanisms are needed to drive efficient regulated exon skipping (Nagengast, 2003).

Somatic inhibition restricts splicing of the Drosophila P-element third intron (IVS3) to
the germ line. This simple system has been exploited to provide a model for a
mechanism of alternative pre-mRNA splicing. Biochemical complementation
experiments reveal that Drosophila somatic extracts inhibit U1 snRNP binding to
the 5' splice site. U1 snRNP binds to a pseudo-5' splice site in the 5' exon and multiprotein complexes bind to an adjacent site. Binding of these factors
appears to mediate the inhibitory effect, because mutations in the pseudo-5' splice
sites block binding and activate splicing in vitro. Likewise, wild-type, but not
mutant, 5' exon RNA titrates inhibitory factors away from the pre-mRNA and
activates splicing. Thus, the pseudo-5' splice sites have been defined as crucial
components of the regulatory element; there is a correlation between inhibitory activity and specific
RNA binding factors from Drosophila somatic cells. The pseudo-5' splice sites also provide a mechanistic
description of somatic inhibition. Because the inhibitory activity involves general
splicing functions such as protein recognition of 5' splice site sequences and changes
in the distribution of bound U1 snRNP, these data may also provide insights into how
splice sites are selected (Siebel, 1992).

Alternative splicing of pre-mRNAs is a versatile regulatory mechanism that can achieve quantitative control of gene expression and functional diversification of gene products. Much progress has been made toward understanding the basic splicing reaction and recognizing exon/intron boundaries, but the mechanisms that regulate alternative splicing are only beginning to be elucidated. Recognition of the 5' splice site by U1 snRNP and of the branchpoint near the 3' splice site by U2 snRNP auxiliary factor (U2AF) are two critical early steps that are regulated in cell- or stage-specific alternative splicing. The picture emerging from biochemical and genetic studies is that splice site selection results from the combined action of conserved consensus sequences that base-pair with the U snRNAs together with protein-protein and protein-RNA interactions that stabilize snRNP binding and mediate bridging interactions between snRNPs at the 5' and 3' splice sites. These interactions involve a growing list of non-snRNP factors, some of which may be responsible for developmental regulation of splice site selection (Burnette, 1999 and references).

Members of the SR family of RNA-binding proteins are required for multiple steps of the splicing reaction in vitro and their concentration can influence splice site competition both in vitro and in overexpression assays using cultured cells. SR proteins are required for the activity of at least some splicing enhancers that stimulate the use of weak 5' or 3' splice sites, and there is evidence for distinct specificities in these interactions. Members of the hnRNP A/B family of RNA-binding proteins also influence splice site selection in a concentration-dependent manner in vitro and when overexpressed in cultured cells. In these assays the hnRNP RNA binding proteins can antagonize the action of SR proteins. These observations have suggested that SR proteins and hnRNP A/B proteins function in vivo as concentration-dependent regulators of alternative splicing. Another possibility is that members of these families serve as cofactors or targets for the actual regulators. Particular SR proteins have been proposed to interact with developmentally specific factors to promote regulation of splicing (Burnette, 1999 and references).

Although a framework of hypotheses is evolving, little is known about regulators of alternative splicing and how they function in vivo. Notable exceptions are Sxl and Tra, proteins that control alternative splicing decisions during sex determination in Drosophila. Because few developmentally specific regulators of alternative splicing have been identified, it is possible that many -- if not most -- alternative splicing decisions are regulated by relatively subtle variations in the levels of general, widely distributed factors, perhaps acting cooperatively or antagonistically as proposed for SR and hnRNP A/B proteins. This is consistent with much correlative evidence and many in vitro observations, but conclusive proof that either type of protein normally regulates an alternative splicing decision in vivo has yet to be obtained. Although null alleles of the Drosophila SR protein gene B52 (homolog of human SRp55) show it to be essential for viability, examination of multiple constitutively and alternatively spliced RNAs has failed to reveal any alterations of splicing even in the absence of detectable protein (Burnette, 1999 and references).

The homeotic gene Ultrabithorax (Ubx) of Drosophila melanogaster was used as a model for regulation of alternative splicing in large and complex transcription units. The six alternative Ubx mRNAs share large protein-coding 5' and 3' exons but differ in the pattern of incorporation of three elements: B is located between two alternative donor sites at the end of the first common exon, whereas mI and mII are internal cassette exons. Within the central nervous system (CNS), different neurons express distinct ratios of Ubx isoforms. The complex and quantitative nature of this regulation is unlike that of other well-studied model systems in Drosophila (e.g., sex-specific splicing in the sex determination hierarchy or germ line-specific splicing of P-element transcripts) but resembles that of many other genes in vertebrates and invertebrates. It seems most likely that this type of alternative splicing is controlled not by highly tissue- and gene-specific splicing regulators but by developmental variations in the concentration or activity of broadly distributed multifunctional factors that may act combinatorially. Hence, Ubx should be a valuable model where genetic approaches can be used to dissect this type of regulation (Burnette, 1999 and references).

Strong reductions of function for the postulated type of regulatory factors would probably cause lethal phenotypes that would be uninterpretable in terms of their effects on Ubx splicing. However, the Ubx splicing pattern should be sensitive to partial reductions in the concentration or activity of these regulatory factors. This may also be true for factors that play important accessory roles in regulation as targets or as constitutively expressed components of regulatory complexes. Two approaches were used to identify such factors. (1) First, a test was carried out to see if the Ubx alternative splicing pattern is altered in heterozygotes for strong loss-of-function mutations. Such mutations are found in a set of genes implicated in the control of alternative splicing in Sxl and P-element RNAs. (2) To identify the location of additional genes involved in regulation of Ubx splicing, a large collection of deficiencies was tested for dominant enhancement of the haploinsufficient Ubx haltere phenotype; it was then asked whether the Ubx splicing pattern is altered in heterozygotes for the interacting deficiencies, and the phenotypic interaction and effect on splicing was traced to specific genes when mutations existed in reasonable candidates. Inclusion of the cassette exons in
Ubx mRNAs is reduced strongly in heterozygotes for hypomorphic alleles of hrp48, which encodes a member of the hnRNP A/B family and is implicated in
control of P-element splicing. Significant reductions of mI and mII inclusion were also observed in heterozygotes for loss-of-function alleles of virilizer, fl(2)d, and
crooked neck. The products of virilizer and fl(2)d are also required for Sxl autoregulation at the level of splicing; crooked neck encodes a protein with structural
similarities to yeast-splicing factors Prp39p and Prp42p. Deletion of at least five other loci caused significant reductions in the inclusion of mI and/or mII (Burnette, 1999).

Coupled RT-PCR assays were used to analyze the pattern of Ubx alternative splicing in heterozygous third instar larvae and in adults. The isoform ratios in third instar larvae were in close agreement with those determined previously using nuclease protection assays. Types Ia and IIa are the predominant Ubx mRNAs and those lacking both mI and mII (isoforms IVa and b) make up only a small fraction of the total. Adults contain a significantly higher proportion of class IV mRNAs than larvae; this differs from previous reports and probably reflects the very early and narrow age distribution of the adults used in this study. It is important to note that the Ubx isoform ratios did not vary significantly between different wild-type strains nor between these and several control strains that carried different balancer chromosomes and irrelevant mutations. These results demonstrate that the mechanism that controls Ubx alternative splicing is robust, a conclusion that is consistent with the faithful conservation of Ubx isoform structure and expression among Drosophila species spanning 60 million years of evolution. The fact that the quantitative isoform pattern revealed by this assay is insensitive to considerable variation in genetic background highlights the significance of the effects described below for specific mutations and deficiencies. Although amplified Ubx cDNA fragments that contain mI but not mII (i.e., hypothetical isoforms IIIa and IIIb) should have the same length as isoforms IIa and IIb, such amplifiers would be expected to exhibit distinctly slower mobility due to the difference in nucleotide sequence (Burnette, 1999).

The products of Sxl, tra, and tra-2 are known regulators of alternative splicing decisions in Drosophila but they are not essential for processes other than sex determination (and dosage compensation, in the case of Sxl) because males that are null for these genes are viable and appear phenotypically normal. However, additional genes [fl(2)d, virilizer, and l(2)49Db] are required for correct control of alternative splicing decisions by Sxl are also essential for viability in both sexes; hence, their products may also have roles in other alternative splicing events. To determine whether these include the control of Ubx alternative splicing, it was asked whether the Ubx isoform ratios are altered in heterozygotes for mutations in these genes. In contrast to the stability described in the preceding section, the Ubx splicing pattern is altered significantly when the expression or function of virilizer or fl(2)d is reduced. The strongest effect is observed with virilizer, using a loss-of-function allele (vir3) that is recessive lethal in both sexes. In heterozygous larvae the proportion of Ubx class I mRNAs declines while that of classes II and IV increases. The proportion of class I that contains the B element is not altered. The increase in classes II and IV indicates that inclusion of both mI and mII is reduced but that the effect on mI exceeds that on mII. Inclusion of mI is also reduced in adults, although the effect was weaker than in larvae. More modest but statistically significant reductions of mI and mII inclusion are also observed in larvae heterozygous for the fl(2)d2 mutation, which is also a loss-of-function allele that is recessive lethal in both sexes (Burnette, 1999).

hrp48 plays a critical role in the inclusion of mI and mII: hrp48 is a member of the hnRNP-A/B family of RNA-binding proteins and forms part of a protein complex that regulates splicing of intron 3 (IVS3) in P-element transcripts. Although repression of IVS3 splicing in somatic tissues is dictated by PSI, which is a soma-specific component of the regulatory complex, the hrp48 protein binds specifically to sequences within the cis-acting regulatory element in the RNA. hrp48 was originally identified as a general component of heterogeneous nuclear ribonucleoprotein particles and the hrp48 gene is essential for viability, so it must perform additional functions unrelated to P-element expression; these functions might include regulation of other splicing decisions. The five known mutant alleles of hrp48 are all P-element insertions in the upstream regulatory region and are not null. Nevertheless, inclusion of mI and mII in Ubx mRNAs is reduced markedly in larvae and adults heterozygous for the strong recessive lethal allele hrp481; weaker alleles, some of which are viable as homozygotes, have similar but more modest effects. The effect of hrp48 mutations resembles that of vir and fl(2) mutations: inclusion of mII is affected more weakly than mI, and the proportion of isoform I that contains the B element is not altered. Heterozygosity for hrp481 reduces inclusion of mI by 27%; this is the strongest effect observed for any mutation or deficiency in this study, indicating that normal levels of hrp48 are critical for inclusion of the internal exons, especially mI, in Ubx mRNAs (Burnette, 1999).

One enhancer, Df(1)64c18g, deletes the genes crooked neck (crn) and kurz (kz), which are located at 2F1 and are both candidate RNA-processing factors. The crn gene encodes a protein with 16 tetratrichopeptide repeats, a motif implicated in protein-protein interactions. Although CRN protein has been proposed to function as a transcription factor involved in cell cycle control, recent data show that it is closely related to the yeast splicing factors Prp39p and Prp42p, which associate with yeast U1 snRNP and are required for splicing. The kz gene encodes a protein with extensive homology to yeast ATP-dependent splicing factors Prp2p, Prp16p, and Prp22p. These proteins define a distinct subfamily of ATP-dependent putative RNA-helicases. Because mutant alleles of these genes are available, a direct test was carried out to see if deletion of one or both might be responsible for enhancement of the Ubx haltere phenotype and whether they affect the Ubx splicing pattern. Like the deficiency, two hypomorphic, recessive lethal alleles of crn (EA130 and RC63) act as dominant zygotic enhancers of Ubx195/+ and Ubx9.22/+. RT-PCR analysis shows that inclusion of mI, but not mII, is reduced significantly in larvae heterozygous for crnEA130. The second allele, crnRC63, has similar effects on the Ubx phenotype and splicing pattern. A recessive lethal allele of kz (DF942) behaves as a weak dominant enhancer of Ubx195/+ and Ubx9.22/+, but RT-PCR analyses does not reveal a significant dominant effect on the Ubx splicing pattern (Burnette, 1999).

The inclusion of mI and mII in Ubx mRNAs is regulated by competition between 5' splice sites that flank each of these exons after they are joined to E5'. As the RNA is transcribed, mI and subsequently mII are spliced constitutively to the upstream exon but can then be removed, together with the downstream intron, using an upstream 5' splice site within E5' or at the junction with this exon. For the majority of nascent RNAs (those initially spliced using 5' splice site a in E5'), a strong 5' splice site is regenerated at the junction between E5' and mI or mII that competes with the mI or mII 5' splice site located 51 nt downstream. For a minority of nascent RNAs (those initially spliced using 5' splice site b in E5') the a site is still present in E5' and can compete with the mI or mII 5' splice site located 78 nt downstream; use of the a site then removes the B element along with mI or mII. Developmental regulation of mI and mII inclusion is achieved by modulating the competition between the upstream and downstream 5' splice sites that flank these exons (Burnette, 1999).

Reduction of function in all of the factors identified in this work leads to reduced inclusion of mI (and in most cases also mII). This suggests roles in suppression of the upstream sites (which strongly match the 5' splice site consensus) or stimulation of the downstream sites (which match the consensus more weakly). It is interesting that three of the factors identified in this study that are required for inclusion of mI and mII in Ubx mRNAs may also be required for suppression of 5' splice site utilization in other RNAs: the functions of virilizer and fl(2)d are required for Sxl to repress splicing of the male-specific exon in its own RNA, and hrp48 is implicated as part of a complex that mediates repression of a 5' splice site in P-element RNA. In addition, heterozygosity for a null allele of sans-fille (snfJ210) does not alter the Ubx splicing pattern, but the antimorphic allele snfe8H, which interferes with autoregulation of Sxl splicing, enhances the Ubx haltere phenotype and increases exclusion of mI and mII (Burnette, 1999 and references).

The products of virilizer, fl(2)d, and snf might function as parts of a complex that mediates active repression of 5' splice site utilization through interactions with U1 snRNP. Formation or stabilization of this repression complex could be directed to different target splice sites through the action of distinct factors that, like Sxl, bind to cis-acting regulatory signals and interact with components of the complex.
An intriguing possibility is that hrp48 interacts (directly or indirectly) with a U1 snRnp/Snf/Vir/Fl(2)D complex to target suppression of splicing at the upstream sites that are used to remove mI. The strong reduction of mI inclusion (27%) observed in hrp481 heterozygotes suggests a critical role for hrp48 in modulating competition between the regenerated and downstream 5' splice sites that flank this exon. Although hrp48 is an hnRNP protein that probably binds nonspecifically to many RNAs, it is also known to form part of a specific complex that blocks use of the 5' splice site for the third intron of P-element RNA in somatic cells. This regulatory complex prevents U1 snRNP from binding at the 5' splice site and recruits it instead, nonproductively, to the more upstream of two overlapping pseudo-5' splice sites within the exon; hrp48 itself makes contact with the downstream pseudo-5' splice site, F2. Splicing of P-element IVS3 in a reporter transgene is partially derepressed in adult escapers homozygous for a semilethal hrp48 allele, indicating that hrp48 is necessary for efficient suppression of the 5' splice site. Hence, it may be significant that a sequence within mI that overlaps the regenerated 5' splice site matches F2 and flanking nucleotides at 8 of 10 positions; this sequence is conserved among four Drosophila species that diverged up to 60 million years but maintain identical regulation of mI inclusion. hrp48 might bind to this sequence and help to recruit U1 snRNP nonproductively to the regenerated 5' splice site at the E5'/mI junction; in intermediates where mI has been spliced to the b site of E5', this complex could also block access to the a site located 27 nt upstream. This would explain why the hrp48, vir, and fl(2)d mutations reduce mI inclusion but do not alter the proportion of class I mRNAs that contain the B element: failure to assemble the repression complex at the E5'/mI junction would allow inappropriate use of both the regenerated site (used to remove mI from E5'a/mI and E5'b/mI intermediates) and the a site (used to remove mI and the B element from E5'b/mI intermediates) (Burnette, 1999 and references).

The effect of hrp48, vir, and fl(2)d mutations on inclusion of exon mII, which does not contain an F2-like element, may not be the result of resplicing at the E5'/mII junction. The reduction of mII inclusion (detected as an increase in class IV mRNAs rather than a decrease in class II) could be explained if the repression complex must remain assembled at the E5'/mI junction to prevent subsequent removal of mI and mII together during splicing of intron 3. Intermediates from which mI is removed during splicing of intron 2 would retain mII. The net result would be an increase in both class II and class IV mRNAs, as observed. In addition, it is noted that the effect of hrp48 mutations on mI and mII inclusion is the opposite of what one would expect from the simple idea that hnRNP A/B proteins generally promote exon skipping (and use of upstream 5' splice sites), antagonizing a general effect of SR proteins that promote exon inclusion (or use of downstream 5' splice sites). The observations presented here are more consistent with a specific role for hrp48 acting through cis-regulatory elements to prevent resplicing of mI. It is more difficult to speculate on the roles of crn or the still-unidentified factors deleted by deficiencies that alter the Ubx splicing pattern. In principle, these could participate in repression of the regenerated 5' splice sites or stimulation of the competing downstream site. They could also be involved in interactions between mI and mII that seem to be required for effective use of the downstream 5' splice site located at the mI/intron 2 boundary. Although a weak homology to the homeodomain led to the proposal that the crooked neck protein functions as a transcription factor, its 16 tetratrichopeptide repeats form a distinct subfamily with those of Prp39p and Prp42p, two splicing factors from yeast that interact with U1 snRNP but appear not to bind RNA directly. A third yeast member of this group has been identified that has more extensive homology to crn ; it will be interesting to learn whether this also functions as a splicing factor (Burnette, 1999 and references).

P-element somatic inhibitor (PSI) is a KH domain-containing splicing factor
highly expressed in Drosophila somatic tissues. A direct
association of PSI with the spliceosomal U1 small nuclear ribonucleoprotein
(snRNP) particle has been detected in somatic nuclear extracts. This interaction is mediated by highly conserved residues within the PSI C-terminal AB motif and the U1
snRNP-specific 70K protein. Through the AB motif, PSI modulates U1 snRNP binding
on the P-element third intron (IVS3) 5' splice site and its upstream exonic
regulatory element. Ectopic expression experiments in the Drosophila female
germline demonstrate that the AB motif also contributes to IVS3 splicing
inhibition in vivo. These data show that the processing of specific target
transcripts, such as the P-element mRNA, is regulated by a functional PSI-U1
snRNP interaction in Drosophila (Labourier, 2001).

Messenger RNAs (mRNAs) in eukaryotic cells are produced from precursor transcripts (pre-mRNAs) by posttranscriptional processing. In metazoans, two processing reactions -- splicing of introns and alternate cleavage and polyadenylation -- are particularly extensive and contribute most significantly to mRNA transcriptome diversity. Splicing is performed by a spliceosome that assembles on each intron and predominantly comprises small nuclear RNPs (snRNPs), U1, U2, U4, and U5, and U6 snRNPs in equal stoichiometry. U1 snRNP (U1) plays an essential role in defining the 5' splice site (5'ss) by RNA:RNA base pairing via U1 snRNA's 5' nine nucleotide (nt) sequence. Using antisense morpholino oligonucleotide complementary to U1 snRNA's 5' end (U1 AMO) that interferes with U1 snRNP's function in human cells, accumulation of introns was observed in many transcripts, as expected for splicing inhibition. However, in addition, the majority of pre-mRNAs terminated prematurely from cryptic polyadenylation signals (PASs) in introns, typically within a short distance from the transcription start site (TSS). These findings indicated that nascent transcripts are vulnerable to premature cleavage and polyadenylation (PCPA) and that U1 has a critical function in protecting pre-mRNA from this potentially destructive process. It was further shown that PCPA suppression is a separate, splicing-independent and U1-specific function, as it does not occur when splicing is inhibited with U2 snRNA AMO or the splicing inhibitor, spliceostatin A (SSA) (Berg, 2012).

These observations were made by transcriptome profiling using partial genome tiling arrays, which provided limited information. In this study, to define the parameters involved in PCPA and its suppression, a strategy (HIDE-seq) was devised to select and sequence only differentially expressed transcripts, identifying changes that occur upon U1 decrease to various levels and in different organisms. The sequence information obtained from HIDE-seq provided genome-wide PCPA maps, and these, together with direct experiments, revealed that U1's PCPA suppression is not only essential for protecting nascent transcripts but is also a global gene expression regulation mechanism. Unexpectedly, PCPA position varied widely with the degree of U1 decrease, trending to usage of more proximal PASs with greater reduction. This yielded mRNAs with shorter 3' untranslated regions (3' UTRs) and alternatively spliced isoforms resulting from usage of more proximal alternative polyadenylation (APA) sites, which is characteristic of activated immune, neuronal, and cancer cells. It was demonstrated that U1 decrease can recapitulate such specific mRNA changes that occur during neuronal activation. Indeed, it was shown that the rapid transcriptional upregulation during neuronal activation is a physiological condition that creates U1 shortage relative to nascent transcripts. Furthermore, U1 overexpression inhibits activated neurons' mRNA signature shortenings. It is suggested that by determining the degree of PCPA suppression, U1 levels play a key role in PAS usage and hence mRNA length. A model is proposed whereby U1 binds to nascent pre-mRNAs cotranscriptionally to explain how U1 shortage results in a corresponding loss of distal PASs suppression from the cleavage and polyadenylation machinery that is associated with the RNA polymerase II (polII) transcription elongation complex (Berg, 2012).